Field of the Invention
The invention related to recombinant vitamin K dependent proteins and methods of preparing the protein in a mammalian cell without the use of heterologous post-translational modification enzymes.
Description of the Related Art
Bleeding disorders can result from a deficiency in the functional levels of one or more of the blood proteins, collectively known as blood coagulation factors, that are required for normal hemostasis, i.e. blood coagulation. The severity of a given bleeding disorder is dependent on the blood level of functional coagulation factors. Mild bleeding disorders are generally observed when the functional level of a given coagulation factor reaches about 5% of normal, but if the functional level falls below 1%, severe bleeding is likely to occur with any injury to the vasculature.
Medical experience has shown that essentially normal hemostasis can be temporarily restored by intravenous infusion of biological preparations containing one or more of the blood coagulation factors. So-called replacement therapy, whereby a biological preparation containing the deficient blood coagulation factor is infused when bleeding occurs (on demand) or to prevent bleeding (prophylactically), has been shown to be effective in managing patients with a wide variety of bleeding disorders. In general, for replacement therapy to be effective, intravenous infusions of the missing coagulation factor are targeted to achieve levels that are well above 5% of normal over a two- to three-day period.
Historically, patients who suffer from hemophilia, a genetically acquired bleeding disorder that results from a deficiency in either blood coagulation Factor VIII (hemophilia A) or Factor IX (hemophilia B), were successfully treated by periodic infusion of whole blood or blood plasma fractions of varying degrees of purity.
More recently, with the advent of biotechnology, biologically active preparations of synthetic (recombinant) blood coagulation factors have become commercially available for treatment of blood coagulation disorders. Recombinant blood coagulation proteins are essentially free of the risks of human pathogen contamination that continue to be a concern that is associated with even high purity commercial preparations that are derived from human blood.
Several of the proteins required for normal blood coagulation are very complex in terms of having multiple structural domains each being associated with a very specific functional property that is essential for the overall effectiveness of the protein in controlling hemostasis and/or preventing thrombosis. In particular, the so-called “vitamin K-dependent” blood coagulation proteins, e.g. Factors II, VII, IX, X, Protein C and Protein S, are very complex proteins and must undergo extensive post-translational modification for normal function. Achieving high levels of functional vitamin K-dependent proteins by recombinant technology has been limited by the structural complexity of these proteins and the inability to create genetically engineered cell systems that overcome the inherent deficiencies in the enzymatic activities required for efficient and complete post-translational modification to occur.
Kaufman, et al, (Kaufman, et al. (1986) The Journal of Biological Chemistry, vol. 261 no, 21: 9622-9628) report the production of recombinant, biologically active Factor IX. However, while upwards of 100 μg/mL of Factor IX was produced, the level of active material was only 1.5%.
Other vitamin K dependent proteins have been produced recombinantly with limited success. Jorgensen, et al. (Jorgensen, et al. (1987) The Journal of Biological Chemistry, vol. 262-(14): 6729-6734) report that human prothrombin was produced in CHO cells at a level of up to 0.55 μg/ml. At this level, the prothrombin was all biologically active. However, when levels were increased 10-15 fold, biological activity dropped to 60%. They hypothesized that the γ-carboxylation system of CHO cells is limited and that only a certain level of protein can be efficiently processed.
Messier, et al. (Messier, et al. (1991) Gene vol. 99: 291-294) cloned and expressed human Factor X in COS-1 Monkey kidney Cells. Both the level produced (0.25-0.27 μg/ml) and the biological activity (9-10%) were low.
Herlitschka, et al. (Herlitschka, et al, (1996) Protein Expression and Purification vol. 8: 358-364) used human prothrombin as a reporter with hygromycin phosphotransferase/dihyrofolate reductase (DHFR) as a dominant selection/amplification fusion marker. Levels of up to 200-250 mU/106 cells/24 hours were produced using 293 kidney cells and 5-15 mU/106 cells/24 hours using CHO cells. Taking 1 Unit as equivalent to about 100 μg, this translates to a maximum level of 25 μg using 293 cells. Although the relative biological activity was not determined, the authors indicated that the 293 cells had been chosen because of its high carboxylation potential.
Himmelspach, et al. (Himmelspach, et al. (2000) Thrombosis Research vol. 97:51-67) obtained 120 μg/mL/day of recombinant human Factor X using DHFR deficient CHO cells with methotrexate selection. Biological activity was up to 25%. The role of Furin in processing of Factor X was investigated by these workers. While Factor X, like Factor IX, also requires gamma carboxylation and post-translational cleavage, it is not clear why higher levels of Factor X having biological activity have been obtained compared to Factor IX. Significant amounts of the recombinant Factor X produced remained covalently attached to the propeptide and/or remained as a single chain precursor. In the presence of recombinant Furin (PACE), the amount of biologically active Factor X approximately doubled (from 22% to 43%). In Factor X, removal of the propeptide appeared to rely upon an endopeptidase other than Furin, while light/heavy chain processing was furin-dependent.
Sun, et al. (Sun, et al. (2005) Blood vol, 106 (12): 3811-3815) reported tht the percentage of carboxylated Factor X can be increased from 50% to 95% by coexpression of Vitamin K epoxide reductase (VKOR).
Wasley, et al. (Wasley, et al. The Journal of Biological Chemistry vol. 268 (12): 8458-8465, 1993) reported that Factor IX is poorly processed in Chinese Hamster Ovary (CHO) cells but that coexpression of PACE (Paired basic Amino acid Cleaving Enzyme) improved processing and specific activity 2-3 fold.
Among the problems encountered in recombinant systems is that in order to produce biologically active Factor IX and Factor VII/VIIa, substantial gamma-carboxylation of glutamic acid residues in the amino terminal region of the protein referred to as the gla-domain, is needed. For example, FVII/VIIa has 10 gla residue sites which should be carboxylated and Factor IX has 12. A majority of these residues must be gamma-carboxylated in order to have bioaetive protein. Additionally, pro-Factor IX, a form of Factor IX that contains a propeptide domain that is required for the efficient intracellular gamma-carboxylation of the protein, must be processed properly prior to secretion, as must Factor VII be processed prior to secretion.
One approach is to co-transfect with genes encoding enzymes which function to post-translationally process Factor IX. Appropriate enzymes include Vitamin K dependent γ-glutamyl carboxylase (VKGC), Vitamin K dependent epoxide reductase (VKOR), and Paired basic amino acid converting enzyme (PACE). U.S. application Ser. No. 11/643,563, filed Dec. 21, 2006 is directed to this approach.
VKGC incorporates a carboxyl group into glutamic acid to modify multiple residues within the vitamin K dependent protein within about 40 residues of the propeptide, within the so-called “gla domain”, VKOR is important for vitamin K dependent proteins because vitamin K is converted to vitamin K epoxide during reactions in which it is a cofactor. The amount of vitamin K in the human diet is limited. Therefore, vitamin K epoxide must be converted back to vitamin K by VKOR to prevent depletion. Consequently, co-transfection with VKOR enhances the appropriate cycling of vitamin K inside the cell and provides sufficient vitamin K for proper functioning of the vitamin K dependent enzymes such as VKGC. The term “PACE” is an acronym for paired basic amino acid converting (or cleaving) enzyme; PACE, is a subtilisin-like endopeptidase, i.e., a propeptide-cleaving enzyme which exhibits specificity for cleavage at basic, residues of a polypeptide, e.g., -Lys-Arg-, -Arg-Arg, or -Lys-Lys-.
While the above mentioned enzymes may be incorporated into a transgenic cell line for processing of vitamin K dependent proteins, mammalian cells naturally produce certain levels of these enzymes endogenously.
The approach taken here is a process of initial selection, whereby a gene, such as a DNA sequence with introns or a cDNA encoding a gene product for a vitamin K dependent protein such as Factor VII or Factor IX is cloned into mammalian cells, followed by selection for transfected clones. The high level expressers are identified, isolated and optionally pooled and may be re-cloned. In any case, the cloned cells are cultured to select even higher expressing clones. The method selects for cell lines which express high levels of vitamin K dependent proteins without requiring co-transfection with multiple heterologous genes, such as genes encoding enzymes necessary for the post-translational modification of vitamin K dependent proteins.